Method for producing retinal tissue and retina-related cells

ABSTRACT

The present invention provides a method for producing a retinal progenitor cell, including (1) a first step of subjecting pluripotent stem cells to floating culture in a serum-free medium to form an aggregate of pluripotent stem cells, and (2) a second step of subjecting the aggregate formed in step (1) to floating culture in a serum-free medium or serum-containing medium each being free of a substance acting on the Sonic hedgehog signal transduction pathway but containing a substance acting on the BMP signal transduction pathway, thereby obtaining an aggregate containing retinal progenitor cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of copending U.S. patentapplication Ser. No. 14/913,628, filed on Feb. 22, 2016, which is theU.S. national phase of International Patent Application No.PCT/JP2014/072065, filed Aug. 22, 2014, which claims the benefit ofJapanese Patent Application No. 2013-173285, filed on Aug. 23, 2013,which are incorporated by reference in their entireties herein.

TECHNICAL FIELD

The present invention relates to a method of producing a retinal tissue,and retina-related cells such as retinal progenitor cell and retinallayer-specific neural cell, and so is on.

BACKGROUND ART

As a method of producing a three-dimensional retinal tissue frompluripotent stem cells, a method of obtaining a multi-layer retinaltissue by forming a homogeneous aggregate of pluripotent stem cells in aserum-free medium, subjecting them to floating culture in the presenceof a basement membrane preparation, and to floating culture in an organculture medium (non-patent document 1 and patent document 1), and amethod of obtaining a multi-layer retinal tissue by forming ahomogeneous aggregate of pluripotent stem cell in a serum-free mediumcontaining a substance inhibiting the Wnt signal pathway, subjectingthem to floating culture in the presence of a basement membranepreparation and floating culture in a serum-containing medium(non-patent document 2 and patent document 2) are shown.

DOCUMENT LIST Patent Documents

patent document 1: WO 2011/055855

patent document 2: WO 2013/077425

non-patent documents

non-patent document 1: Nature, 472, 51-56 (2011)

non-patent document 2: Cell Stem Cell, 10(6), 771-785 (2012)

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

The development of a production method of a retinal tissue from apluripotent stem cell has been desired.

Means of Solving the Problems

The present invention provides a method of producing a retinal tissue,and retina-related cells such as retinal progenitor cell and retinallayer-specific neural cell from pluripotent stem cells, and so on.

Accordingly, the present invention provides:

[1] a method for producing a retinal progenitor cell, comprising(1) a first step of subjecting pluripotent stem cells to floatingculture in a serum-free medium to form an aggregate of pluripotent stemcells, and(2) a second step of subjecting the aggregate formed in step(1) to floating culture in a serum-free medium or serum-containingmedium each being free of a substance acting on the Sonic hedgehogsignal transduction pathway and containing a substance acting on the BMPsignal transduction pathway, thereby obtaining an aggregate containingretinal progenitor cells (hereinafter sometimes to be indicated asproduction method 1 of the present invention);a method for producing a retinal tissue, comprising(1) a first step of subjecting pluripotent stem cells to floatingculture in a serum-free medium to form an aggregate of pluripotent stemcells,(2) a second step of subjecting the aggregate formed in step (1) tofloating culture in a serum-free medium or serum-containing medium eachbeing free of a substance acting on the Sonic hedgehog signaltransduction pathway and containing a substance acting on the BMP signaltransduction pathway, thereby obtaining an aggregate containing retinalprogenitor cells, and(3) a third step of subjecting the aggregate formed in step (2) tofloating culture in a serum-free medium or serum-containing medium eachbeing free of any of a substance acting on the Sonic hedgehog signaltransduction pathway, a substance acting on the BMP signal transductionpathway and a substance acting on the Wnt signal pathway, therebyobtaining an aggregate containing retinal tissues and beingsubstantially free of non-neural head ectoderm (hereinafter sometimes tobe indicated as production method 2 of the present invention);[3] a method for producing a retinal layer-specific neural cell,comprising(1) a first step of subjecting pluripotent stem cells to floatingculture in a serum-free medium to form an aggregate of pluripotent stemcells,(2) a second step of subjecting the aggregate formed in step (1) tofloating culture in a serum-free medium or serum-containing medium eachbeing free of a substance acting on the Sonic hedgehog signaltransduction pathway and containing a substance acting on the BMP signaltransduction pathway, thereby obtaining an aggregate containing retinalprogenitor cells, and(3) a third step of subjecting the aggregate formed in step (2) tofloating culture in a serum-free medium or serum-containing medium eachbeing free of any of a substance acting on the Sonic hedgehog signaltransduction pathway, a substance acting on the BMP signal transductionpathway and a substance acting on the Wnt signal pathway until theintended retinal layer-specific neural cells appear, thereby obtainingan aggregate containing retinal tissues containing the intended retinallayer-specific neural cells and being substantially free of non-neuralhead ectoderm (hereinafter sometimes to be indicated as productionmethod 3 of the present invention);[4] the method of any of the aforementioned [1] to [3], wherein thepluripotent stem cells are primate pluripotent stem cells;[5] the method of any of the aforementioned [1] to [4], wherein thepluripotent stem cells are human pluripotent stem is cells;[6] the method of any of the aforementioned [1] to [5], wherein the step(1) and step (2) are performed in the presence of a serum replacement;[7] the method of any of the aforementioned [1] to [6], wherein thefloating culture is performed in the absence of a basement membranepreparation;[8] the method of any of the aforementioned [1] to [7], wherein thesubstance acting on the BMP signal transduction pathway is one or moreproteins selected from the group consisting of BMP2, BMP4, BMP7 andGDF7;[9] the method of any of the aforementioned [1] to [8], wherein thesubstance acting on the BMP signal transduction pathway is added to themedium between day 1 and day 15 from the start of the floating culturein step (1);[10] a reagent for evaluating toxicity or drug efficacy, comprising aretinal progenitor cell, retinal tissue or retinal layer-specific neuralcell, the cell or tissue being produced by the method of any of theaforementioned [1] to [9];

[11] a method of evaluating toxicity or drug efficacy of a testsubstance, comprising bringing a retinal progenitor cell, retinal tissueor retinal layer-specific neural cell, the cell or tissue being producedby the method of any of the aforementioned [1] to [9], into contact withthe test substance, and examining the influence of the substance on thecell or tissue;

[12] a therapeutic agent for a disease due to a disorder of a retinaltissue, comprising a retinal progenitor cell, a retinal tissue or aretinal layer-specific neural cell, the cell or tissue being produced bythe method of any of the aforementioned [1] to [9];[13] a method of treating a disease due to a disorder of a retinaltissue, comprising transplanting an effective amount of a retinalprogenitor cell, a retinal tissue or a retinal layer-specific neuralcell, the cell or tissue being produced by the method of any of theaforementioned [1] to [9], to a subject in need of the transplantation;[14] a retinal progenitor cell, a retinal tissue or a retinallayer-specific neural cell, the cell or tissue being produced by themethod of any of the aforementioned [1] to [9], for use in the treatmentof a disease due to a disorder of a retinal tissue; and so on.

Effect of the Invention

According to the production method of the present invention, a retinalprogenitor cell, a retinal tissue or a retinal layer-specific neuralcell can be produced with high efficiency. In the production method ofthe present invention, since a retinal progenitor cell, a retinal tissueor a retinal layer-specific neural cell can be obtained by floatingculture of an aggregate without adding a basement membrane preparationto a medium, namely, in the absence of a basement membrane preparation,the risk of contamination of the obtained cell or tissue with acomponent derived from a heterologous species is reduced. According tothe production method of the present invention, a retinal tissue, orretina-related cells such as retinal progenitor cell and retinallayer-specific neural cell can be efficiently provided for the purposeof toxicity or efficacy evaluation of a chemical substance etc., atransplantation treatment and so on.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows light field image (A) and fluorescence image (B) at day 18from the start of floating culture of aggregates, derived from RAX::GFPknock-in human embryonic stem cells, without adding a substance actingon the BMP signal transduction pathway to the medium, light field image(C) and fluorescence image (D) at day 18 from the start of floatingculture of aggregates, derived from RAX::GFP knock-in human embryonicstem cells, in a medium supplemented with BMP2 to 100 ng/ml at day 3from the start of the floating culture, light field image (E) andfluorescence image (F) at day 18 from the start of floating culture ofaggregates, derived from RAX::GFP knock-in human embryonic stem cells,in a medium supplemented with BMP4 at 1.5 nM at day 3 from the start ofthe floating culture, light field image (G) and fluorescence image (H)at day 18 from the start of floating culture of aggregates, derived fromRAX::GFP knock-in human embryonic stem cells, in a medium supplementedwith BMP7 at 100 ng/ml at day 3 from the start of the floating culture,and light field image (I) and fluorescence image (J) at day 18 from thestart of floating culture of aggregates, derived from dRAX::GFP knock-inhuman embryonic stem cells, in a medium supplemented with GDF7 at 100ng/ml at day 3 from the start of the floating culture.

FIG. 2 shows light field image (A), fluorescence image (B) and FACShistogram (C) at day 26 from the start of floating culture ofaggregates, derived from RAX::GFP knock-in human embryonic stem cells,without adding a substance acting on the BMP signal transduction pathwayto the medium, light field image (D) and fluorescence image (E) and FACShistogram (F) at day 26 from the start of floating culture ofaggregates, derived from RAX::GFP knock-in human embryonic stem cells,in a medium supplemented with BMP4 to 1.5 nM simultaneously with thestart of the floating culture (day 0), light field image (G),fluorescence image (H) and FACS histogram (I) at day 26 from the startof floating culture of aggregates, derived from RAX::GFP knock-in humanembryonic stem cells, in a medium supplemented with BMP4 to 1.5 nM atday 1 from the start of is the floating culture, light field image (J),fluorescence image (K) and FACS histogram (L) at day 26 from the startof floating culture of aggregates, derived from RAX::GFP knock-in humanembryonic stem cells, in a medium supplemented with BMP4 to 1.5 nM atday 2 from the start of the floating culture, and light field image (M),fluorescence image (N) and FACS histogram (0) at day 26 from the startof floating culture of aggregates, derived from RAX::GFP knock-in humanembryonic stem cells, in a medium supplemented with BMP4 at 1.5 nM atday 3 from the start of the floating culture.

FIG. 3 shows light field image (A) and fluorescence image (B) at day 24from the start of floating culture of aggregates, derived from RAX::GFPknock-in human embryonic stem cells, without adding a substance actingon the BMP4 signal transduction pathway to the medium, light field image(C) and fluorescence image (D) at day 24 from the start of floatingculture of aggregates, derived from RAX::GFP knock-in human embryonicstem cells, in a medium supplemented with BMP4 to 1.5 nM at day 6 fromthe start of the floating culture, light field image (E) andfluorescence image (F) at day 24 from the start of floating culture ofaggregates, derived from RAX::GFP knock-in human embryonic stem cells,in a medium supplemented with BMP4 at 1.5 nM at day 9 from the start ofthe floating culture, light field image (G) and fluorescence image (H)at day 24 from the start of floating culture of aggregates, derived fromRAX::GFP knock-in human embryonic stem cells, in a medium supplementedwith BMP4 at 1.5 nM at day 12 from the start of the floating culture,and light field image (I) and fluorescence image (J) at day 24 from thestart of floating culture of aggregates, derived from dRAX::GFP knock-inhuman embryonic stem cells, in a medium supplemented with BMP4 at 1.5 nMat day 15 from the start of the floating culture.

FIG. 4 shows GFP fluorescence image (A), fluorescence immunostainedimage (B) using an anti-Chx10 antibody and Hoechst stained image (C) ofcryosections of aggregates, derived from RAX::GFP knock-in humanembryonic stem cells, which were subjected to floating culture up to day26 from the start of the floating culture in a medium supplemented withBMP4 at 1.5 nM at day 3 from the start of the floating culture.

FIG. 5 shows immunostained image (A) using an anti-Recoverin antibody,immunostained image (B) using an anti-Nrl antibody, immunostained image(C) using an anti-RXR-gamma antibody, immunostained image (D) using ananti-Chx10 antibody, immunostained image (E) using an anti-Calretininantibody, and immunostained image (F) using an anti-Calbindin antibodyof cryosections of aggregates, derived from RAX::GFP knock-in humanembryonic stem cells, which were subjected to floating culture up to day117 from the start of the floating culture in a medium supplemented withBMP4 at 1.5 nM at day 3 from the start of the floating culture.

FIG. 6 shows immunostained image (A) using an anti-Chx1O antibody,immunostained image (B) using an anti-Pax6 antibody, immunostained image(C) using an anti-Crx antibody, and immunostained image (D) using ananti-Brn3b antibody of cryosections of aggregates, derived from RAX::GFPknock-in human embryonic stem cells, which were subjected to floatingculture up to day 50 from the start of the floating culture in s amedium supplemented with BMP4 at 1.5 nM at day 6 from the start of thefloating culture.

DESCRIPTION OF EMBODIMENTS

Mode(s) for carrying out the present invention is explained in detailbelow.

The “vector” in the present invention means a vector capable oftransferring a desired polynucleotide sequence into an intended cell.Examples of such vector include a vector capable of autonomouslyreplicating in a host cell such as prokaryotic cell, yeast, animal cell,plant cell, insect cell, animal individual and plant individual, avector capable of being incorporated into a chromosome of a host cell, avector containing a promoter at a position suitable for polynucleotidetranscription, and so on.

Of such vectors, a vector suitable for cloning is sometimes indicated asa “cloning vector”. Examples of the cloning vector include a vectorgenerally having multiple cloning sites containing a plurality ofrestriction enzyme sites. For example, the vectors described in“Molecular Cloning (3rd edition)” by Sambrook, J and Russell, D. W.,Appendix 3 (Volume 3), Vectors and Bacterial strains. A3.2 (Cold SpringHarbor USA, 2001)) can be mentioned.

The “vector” in the present invention also includes “expression vector”and “reporter vector”. In the “expression vector”, various regulatoryelements in addition to a structural gene and a promoter that regulatesthe expression thereof may be linked in such a manner that they can beoperable in the host cell. In the “reporter vector”, various regulatoryelements in addition to a reporter gene and a promoter that regulatesthe expression thereof may be linked in such a manner that they can beoperable in the host cell. Examples of the “regulatory element” includeterminator and enhancer. The “expression vector” and “reporter vector”may further include selection marker genes such as drug resistance gene.

Examples of the “cloning vector” include (a) lambda FIX vector, which isa phage vector, for the construction of a genomic library, (b) lambdaZAP vector, which is a phage vector, for the construction of a cDNAlibrary, and (c) plasmid vectors such as pBluescript II SK+/−, pGEM, andpCR2.1 vector, for cloning of genomic DNA. Examples of the “expressionvector” include plasmid vectors such as pSV2/neo vector, pcDNA vector,pUC18 vector, pUC19 vector, pRc/RSV vector, pLenti6/V5-Dest vector,pAd/CMV/V5-DEST vector, pDON-AI-2/neo vector, and pMEI-5/neo vector.Examples of the “reporter vector” include pGL2 vector, pGL3 vector,pGL4.10 vector, pGL4.11 vector, pGL4.12 vector, pGL4.70 vector, pGL4.71vector, pGL4.72 vector, pSLG vector, pSLO vector, pSLR vector, pEGFPvector, pAcGFP vector, and pDsRed vector. These vectors can be utilizedas appropriate by reference to the aforementioned Molecular Cloningreference.

As a technique for introducing a nucleic acid molecule into a cell, forexample, transformation, transduction, transfection and so on can bementioned. As such introduction technique, for example, the methodsdescribed in Ausubel F. A. et al. ed. (1988), Current Protocols inMolecular Biology, Wiley, New York, NY; Sambrook J. et al. (1987),Molecular Cloning: A Laboratory Manual, 2nd Ed. and 3rd Ed.; Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.; Sambrook J. et al.(2001) Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.; extra issue,Experimental Medicine “transgene & expression analysis experimentmethod” YODOSHA CO., LTD., 1997, and so on can be specificallymentioned. As the technique for confirming intracellular introduction ofa gene, for example, Northern blot analysis or Western blot analysis canbe mentioned.

The “floating culture” in the present invention means cultivating underconditions prohibiting adhesion of cell or cell mass to a cell culturevessel material etc.

The cell culture vessel to be used in floating culture is notparticularly limited as long as it enables “floating culture”, and thoseof ordinary skill in the art can appropriately determine same. Examplesof such cell culture vessel include flask, tissue culture flask, dish,petri dish, tissue culture dish, multidish, microplate, microwell plate,micropore, multiplate, multiwell plate, chamber slide, schale, tube,tray, culture bag, and roller bottle. Since these cell culture vesselsare used for floating culture, they are preferably cell non-adhesive. Asa cell non-adhesive vessel, one having its surface not artificiallytreated to improve cell adhesiveness (e.g., coating treatment withextracellular matrix, etc.) and so on can be used.

The medium to be generally used in the present invention can be preparedfrom a medium used for culture of animal cell as a basal medium.Examples of the basal medium include those that can be used forculturing animal cells, such as BME medium, BGJb medium, CMRL1066medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDMmedium, Medium199 medium, Eagle MEM medium, aMEM medium, DMEM medium,F-12 medium, Ham's medium, RPMI1640 medium, Fischer's medium, and mixedmedium thereof.

The “serum-free medium” in the present invention means a medium free ofunadjusted or unpurified serum. In the present invention, a mediumcontaining purified blood-derived components and animal tissue-derivedcomponents (e.g., growth factor) is considered to be a serum-free mediumunless unadjusted or unpurified serum is contained therein.

The serum-free medium may contain a serum replacement. Examples of theserum replacement include those appropriately containing, for example,albumin, transferrin, fatty acid, collagen precursor, trace element,2-mercaptoethanol or 3′ thiolglycerol, an equivalent thereof and so on.Such serum replacement can be prepared by, for example, the methoddescribed in WO98/30679. In addition, the serum replacement can be acommercially available product. Examples of such commercially availableserum replacement include KNOCKOUT™ Serum Replacement (manufactured byInvitrogen: hereinafter sometimes to be also indicated as KSR),Chemically defined lipid concentrate (manufactured by Gibco), andGLUTAMAX™ supplement (manufactured by Gibco).

The serum-free medium to be used for floating culture may contain fattyacid or lipid, amino acid (e.g., non-essential amino acid), vitamin,growth factor, cytokine, antioxidant, 2-mercaptoethanol, pyruvic acid,buffering agent, inorganic salts and so on.

To avoid complicated preparation, a serum-free medium supplemented withan appropriate amount (e.g., about 1-about 20%) of commerciallyavailable KSR can be used as the serum-free medium (e.g., mediumobtained by adding 10% KSR and 450 μM 1-monothioglycerol to a 1:1mixture of F-12 medium and IMDM medium).

The “serum-containing medium” in the present invention s means a mediumcontaining unadjusted or unpurified serum. The medium may contain fattyacid or lipid, amino acid (e.g., non-essential amino acid), vitamin,growth factor, cytokine, antioxidant, 2-mercaptoethanol,1-monothioglycerol, pyruvic acid, buffering agent, inorganic salts andso on.

The “basement membrane preparation” in the present invention refers toone containing basement membrane-constituting components having afunction to control cell form, differentiation, growth, motility,expression of function and so on which are similar to those ofepithelial cell, when intended cells capable of forming a basementmembrane are plated thereon and cultured. Here, the “basement membraneconstituting component” refers to an extracellular matrix molecule inthe form of a thin membrane present between epithelial cell layer andinterstitial cell layer and so on in animal tissues. A basement membranepreparation can be produced by, for example, removing cells capable offorming a basement membrane, which adhere onto a support via a basementmembrane, with a solution capable of dissolving the lipid of the cells,an alkali solution and so on. Examples of preferable basement membranepreparation include products commercially available as basement membranecomponents (e.g., MATRIGEL™ extracellular matrix (manufactured byBeckton Dickinson)), and extracellular matrix molecules known asbasement membrane components (e.g., laminin, type IV collagen, heparansulfate proteoglycan, entactin and so on).

MATRIGEL™ extracellular matrix is a product extracted from a basementmembrane derived from Engelbreth Holm Swarn (EHS) mouse sarcoma. Themain component of MATRIGEL™ extracellular matrix is type IV collagen,laminin, heparan sulfate proteoglycan, and entactin. In addition tothese, TGF-β, fibroblast growth factor (FGF), tissue plasminogenactivator, and a growth factor naturally produced by EHS tumor arecontained. The “growth factor reduced product” of MATRIGEL™extracellular matrix has a lower growth factor concentration than commonMATRIGEL™ extracellular matrix, and the standard concentration thereofis <0.5 ng/ml for EGF, <0.2 ng/ml for NGF, <5 pg/ml for PDGF, 5 ng/mlfor IGF-1, and 1.7 ng/ml for TGF-β.

The “medium containing substance X” in the present invention means amedium supplemented with an exogeneous substance X or a mediumcontaining an exogenous substance X, and the “medium free of substanceX” means a medium not supplemented with an exogenous substance X or amedium not containing an exogenous substance X. Here, the “exogenoussubstance X” means a substance X exogeneous to a cell or tissue to becultured in the medium, and an endogenous substance X produced by thecell or tissue is not included therein.

For example, a “medium containing a substance acting on the BMP signaltransduction pathway” is a medium supplemented with an exogenoussubstance acting on the BMP signal transduction pathway or a mediumcontaining an exogenous substance acting on the BMP signal transductionpathway. A “medium free of a substance acting on the Sonic hedgehogsignal transduction pathway” is a medium not supplemented with anexogenous substance acting on the Sonic hedgehog signal transductionpathway or a medium not containing an exogenous substance acting on theSonic hedgehog signal transduction pathway.

The “primates” in the present invention mean mammals belonging toprimate. Examples of the primates include Strepsirrhini such as lemur,loris, and Tsubai, and Haplorhini such as monkey, anthropoid ape, andhuman.

In the present invention, the “stem cell” refers to a cell thatmaintains the same differentiation capacity even after cell division,which can contribute to the regeneration of a tissue thereof when thetissue is injured. Here, the stem cell may be an embryonic stem cell(hereinafter sometimes to be referred to as ES cell) or a tissue stemcell (also called tissular stem cell, tissue-specific stem cell orsomatic stem cell), or an artificial pluripotent stem cell (iPS cell:induced pluripotent stem cell). As is appreciated from the fact that theabove-mentioned stem cell-derived tissue cell can regenerate a tissue,it is known that the stem cell can differentiate into a normal cellclose to one in a living body.

Stem cells are available from given organizations, or a commerciallyavailable product can also be purchased. For example, human embryonicstem cells, KhES-1, KhES-2 and KhES-3, are available from KyotoUniversity's Institute for Frontier Medical Sciences. EB5 cell isavailable from RIKEN, and D3 cell line is available from ATCC, each ofwhich is a mouse embryonic stem cell.

Stem cells can be maintained by culturing according to a method knownper se. For example, human stem cells can be maintained by culturing ina medium supplemented with KNOCKOUT™ Serum Replacement (Invitrogen).Mouse stem cells can be maintained by adding fetal calf serum (FCS) andLeukemia Inhibitory Factor (LIF) and culturing without feeder cells.

In the present invention, the “pluripotent stem cell” refers to a stemcell that can be cultured in vitro and has an ability to differentiateinto any cell (triploblast (ectoderm, mesoderm, endoderm)-derivedtissue) constituting a living body except for placenta (pluripotency),and an embryonic stem cell (ES cell) is included in the pluripotent stemcell. The “pluripotent stem cell” is obtained from fertilized egg, cloneembryo, reproductive stem cell, and stem cell in a tissue. A cell havingartificial differentiation pluripotency similar to that of embryonicstem cells, after introducing several kinds of genes into a somatic cell(also called artificial pluripotent stem cell) is also included in thepluripotent stem cell. Pluripotent stem cell can be produced by a methodknown per se. Examples of the production method include the methodsdescribed in Cell, 2007, 131(5) pp. 861-872 and Cell, 2006, 126(4) pp.663-676.

In the present invention, the “embryonic stem cell (ES cell)” refers toa stem cell having a self replication ability and multipotency(particularly, “pluripotency”), which is a pluripotent stem cell derivedfrom an early embryo. Embryonic stem cell was first established in 1981,and has also been applied to the generation of knockout mouse since1989. In 1998, a human embryonic stem cell was established, which isalso being utilized for regenerative medicine.

In the present invention, the “artificial pluripotent stem cell” refersto a cell induced to have multipotency by directly reprogramming adifferentiated cell such as fibroblast etc. by the expression of severalkinds of genes such as Oct3/4, Sox2, Klf4, and Myc, which wasestablished by Yamanaka et al. in mouse cell in 2006 (Cell. 2006,126(4), pp. 663-676). In 2007, induced pluripotent stem cell was alsoestablished in human fibroblast, and has multipotency similar to that ofembryonic stem cells (Cell, 2007, 131(5) pp. 861-872; Science, 2007,318(5858) pp. 1917-1920; Nat. Biotechnol., 2008, 26(1) pp. 101-106).

A genetically-modified pluripotent stem cell can be produced, forexample, using a homologous recombination technique. Examples of thegene on the chromosome to be modified include a cell marker gene, ahistocompatibility antigen gene, a gene related to a disease due to adisorder of nerve system cell and so on. A target gene on the chromosomecan be modified by the methods described in Manipulating the MouseEmbryo, A Laboratory Manual, Second Edition, Cold Spring HarborLaboratory Press (1994); Gene Targeting, A Practical Approach, IRL Pressat Oxford University Press (1993); Bio Manual series 8, gene targeting,Production of mutant mouse by using ES cells, YODOSHA CO., LTD. (1995)and so on.

To be specific, for example, the genomic gene of a target gene to bemodified (e.g., cell marker gene, histocompatibility antigen gene,disease-related gene and so on) is isolated, and a target vector usedfor homologous recombination of the target gene is produced using theisolated genomic gene. The produced target vector is introduced intostem cells, and cells showing homologous recombination between thetarget gene and the target vector are selected, whereby stem cellshaving modified gene on the chromosome can be produced.

As a method for isolating the genomic gene of the target gene, knownmethods described in Molecular Cloning, A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press (1989), Current Protocolsin Molecular Biology, John Wiley & Sons (1987-1997) and so on can bementioned. Moreover, the genomic gene of the target gene can be isolatedusing genomic DNA library screening system (manufactured by GenomeSystems), Universal GenomeWalker Kits (manufactured by CLONTECH) and soon.

A target vector used for homologous recombination of the target gene canbe produced, and a homologous recombinant can be efficiently selectedaccording to the methods described in Gene Targeting, A PracticalApproach, IRL Press at Oxford University Press (1993); Bio Manual series8, gene targeting,

Production of mutant mouse by using ES cells, YODOSHA CO., LTD. (1995)and so on. The target vector may be any of replacement type andinsertion type, and the selection method may be positive selection,promoter selection, negative selection, polyA selection and so on.

As a method for selecting an intended homologous recombinant from theselected cell lines, Southern hybridization method, PCR method and so onfor genomic DNA can be mentioned.

The “aggregate” in the present invention refers to a mass of the cellsdispersed in the medium but gathered to form same. The “aggregate” inthe present invention includes an aggregate formed by the cellsdispersed at the start of the floating culture and an aggregate alreadyformed at the start of the floating culture.

When cells are gathered to form cell aggregates and the aggregates aresubjected to floating culture, to “form aggregate” means to “rapidlyaggregate a given number of dispersed cells” to form qualitativelyhomogeneous cell aggregates.

In the present invention, it is preferable to rapidly gather pluripotentstem cells to allow for formation of an aggregate of pluripotent stemcells. By forming an aggregate of pluripotent stem cells in this manner,an epithelium-like structure can be formed with good reproducibility inthe cells induced to differentiate from the formed aggregate.

Examples of the experimental operation to form an aggregate include amethod involving keeping cells in a small space by using a plate withsmall wells (96 well plate), micropore and so on, a method involvingaggregating cells by centrifugation for a short time using a smallcentrifugation tube.

Whether aggregates of pluripotent stem cells have been formed andwhether an epithelial-like structure has been formed in the cellsforming the aggregate can be determined based on the size and cellnumber of aggregates, macroscopic morphology, microscopic morphology bytissue staining analysis and uniformity thereof, expression ofdifferentiation and undifferentiation markers and uniformity thereof,control of expression of differentiation marker and synchronism thereof,reproducibility of differentiation efficiency between aggregates, and soon.

In the present invention, the “tissue” refers to a structure of a cellpopulation, which has a conformation wherein more than one type of celldifferent in the shape and property are sterically configured in a givenpattern.

In the present invention, the “retinal tissue” means a retinal tissuewherein at least two or more types of cells such as photoreceptors,horizontal cells, bipolar cells, amacrin cells, retinal ganglion cells,their precursor cells or retinal progenitor cells thereof, whichconstitute respective retinal layers in living retina, are stericallyarranged in layers. With regard to each cell, which cell constituteswhich retinal layer can be confirmed by a known method, for example, thepresence or absence or the level of the expression of a cell marker andthe like.

The “retinal layer” in the present invention means each layerconstituting the retina. Specific examples thereof include retinalpigment epithelial layer, photoreceptor layer, external limitingmembrane, outer nuclear layer, outer plexiform layer, inner nuclearlayer, inner plexiform layer, ganglion cell layer, nerve fiber layer andinner limiting membrane.

The “retinal layer-specific neural cell” in the present invention meansa neural cell constituting a retinal layer and specific to the retinallayer. Examples of the retinal layer-specific neural cell includebipolar cell, ganglion cell, amacrine cell, horizontal cell,photoreceptor, pigment epithelium cell, rod cell and cone cell.

The “retinal progenitor cell” in the present invention refers to aprogenitor cell that can be differentiated into any mature retinal cellof a photoreceptor, a horizontal cell, a bipolar cell, an amacrine cell,a retinal ganglion cell and a retinal pigment epithelial cell.

The photoreceptor precursor cell, horizontal precursor cell, bipolarprecursor cell, amacrine precursor cell, retinal ganglion precursor celland retinal pigment epithelial precursor cell are precursor cellsdetermined to differentiate into a photoreceptor, a horizontal cell, abipolar cell, an amacrine cell, a retinal ganglion cell, and a retinalpigment epithelial cell, respectively.

Examples of the retinal cell marker include Rax and PAX6 expressed inretinal progenitor cells, Nkx2.1 expressed in progenitor cells ofhypothalamus neuron but not expressed in retinal progenitor cells, Sox1expressed in hypothalamus neuroepithelium and not expressed in retina,Crx expressed in precursor cells of photoreceptor and the like. Examplesof the retinal layer-specific neural cell marker include Chx10 and L7expressed in bipolar cells, Tuj1 and Brn3 expressed in ganglion cells,Calretinin expressed in amacrine cells, Calbindin expressed inhorizontal cells, Rhodopsin and Recoverin expressed in photoreceptors,RPE65 and Mitf expressed in pigment epithelium cells, Nrl expressed inrod cells, Rxr-gamma expressed in cone cells and the like.

The production method 1 of the present invention is a method forproducing retinal progenitor cells, which includes the following steps(1) and (2):

(1) a first step of subjecting pluripotent stem cells to floatingculture in a serum-free medium to form an aggregate of pluripotent stemcells, and(2) a second step of subjecting the aggregate formed in step (1) tofloating culture in a serum-free medium or serum-containing medium eachbeing free of a substance acting on the Sonic hedgehog signaltransduction pathway and containing a substance acting on the BMP signaltransduction pathway, thereby obtaining an aggregate containing retinalprogenitor cells.

Step (1) for subjecting pluripotent stem cells to floating culture in aserum-free medium to form an aggregate of pluripotent stem cells isexplained.

The serum-free medium used in step (1) is not particularly limited aslong as it is as mentioned above. For example, a serum-free medium notsupplemented with any of a substance acting on the BMP signaltransduction pathway and a substance inhibiting the Wnt signal pathwaycan be used. To avoid complicated formulation process, a serum-freemedium supplemented with an appropriate amount of a serum replacementsuch as commercially available KSR (e.g., a medium obtained by adding10% KSR, 450 μM 1-monothioglycerol and 1×Chemically Defined LipidConcentrate to a 1:1 mixture of IMDM and F-12) is preferably used. Theamount of KSR to be added to a serum-free medium is generally about 1%to about 20%, preferably about 2% to about 20%, in the case of, forexample, human ES cells.

The culture conditions such as culture temperature, CO₂ concentration instep (1) can be appropriately determined. The culture temperature is,for example, about 30° C. to about 40° C., preferably about 37° C. TheCO₂ concentration is, for example, about 1 to about 10%, preferablyabout 5%.

The concentration of the pluripotent stem cells in step (1) can bedetermined as appropriate to form aggregates of pluripotent stem cellsmore uniformly and efficiently. For example, when human ES cells aresubjected to floating culture using a 96 well microwell plate, a liquidprepared to about 1×10³ to about 5×10⁵ cells, preferably about 3×10³ toabout 5×10⁴ cells, more preferably about 5×10³ to about 3×10⁴ cells,most preferably about 1.2×10⁴ cells, per well is added to a well, andthe plate is left standing to form aggregates.

The time of floating culture necessary for forming aggregates can bedetermined as appropriate according to the pluripotent stem cell to beused, to allow for uniform aggregation of the cells. To form uniformaggregates, it is desirably as short as possible. For example, in thecase of human ES cells, aggregates are formed preferably within about 24hr, more preferably within about 12 hr. The time for aggregate formationcan be appropriately adjusted by controlling the tools for aggregatingthe cells, centrifugation conditions and so on.

Whether aggregates of pluripotent stem cells have been formed can bedetermined based on the size and cell number of aggregates, macroscopicmorphology, microscopic morphology by tissue staining analysis anduniformity thereof, expression of differentiation and undifferentiationmarkers and uniformity thereof, control of expression of differentiationmarker and synchronism thereof, reproducibility of differentiationefficiency between aggregates, and so on.

Step (2) including subjecting the aggregate formed in step (1) tofloating culture in a serum-free medium or serum-containing medium eachbeing free of a substance acting on the Sonic hedgehog signaltransduction pathway and containing a substance acting on the BMP signaltransduction pathway, thereby obtaining an aggregate containing retinalprogenitor cells is explained.

As the medium to be used in step (2), for example, a serum-free mediumor a serum-containing medium not supplemented with a substance acting onthe Sonic hedgehog signal transduction pathway and supplemented with asubstance acting on the BMP signal transduction pathway is used, andaddition of a basement membrane preparation is not necessary.

The serum-free medium or serum-containing medium to be used as suchmedium is not particularly limited as long as it is as mentioned above.To avoid complicated formulation process, a serum-free mediumsupplemented with an appropriate amount of a serum replacement such ascommercially available KSR (e.g., a medium obtained by adding 10% KSR,450 μM 1-monothioglycerol and 1×Chemically Defined Lipid Concentrate toa 1:1 mixture of IMDM and F-12) is preferably used. The amount of KSR tobe added to a serum-free medium is generally about 1% to about 20%,preferably about 2% to about 20%, in the case of, for example, human EScells.

As the serum-free medium to be used in step (2), the serum-free mediumused in step (1) may be used as it is as s long as it does not contain asubstance acting on the Sonic hedgehog signal transduction pathway, ormay be replaced with a fresh serum-free medium. When the serum-freemedium used in step (1), which does not contain a BMP signaltransduction pathway substance, is directly used for step (2), asubstance acting on the BMP signal transduction pathway only needs to beadded to the medium.

A substance acting on the Sonic hedgehog (hereinafter sometimes to beindicated as Shh) signal transduction pathway is a substance that canenhance signal transduction mediated by Shh. Examples of the substanceacting on the Shh signal transduction pathway include proteins belongingto the Hedgehog family (e.g., Shh), Shh receptor, Shh receptor agonist,Purmorphamine, SAG and so on.

A medium free of “a substance acting on the Sonic hedgehog signaltransduction pathway” also includes a medium substantially free of asubstance acting on the Sonic hedgehog signal transduction pathway, suchas a medium free of a substance acting on the Sonic hedgehog signaltransduction pathway, at a concentration exerting an adverse influenceon the selective differentiation into retinal progenitor cell andretinal tissue.

A substance acting on the BMP signal transduction pathway is a substancethat can enhance signal transduction pathway mediated by BMP. Examplesof the substance acting on the BMP signal transduction pathway includeBMP proteins such as BMP2, BMP4 or BMP7, GDF proteins such as GDF7,anti-BMP receptor antibody, BMP partial peptide and so on. BMP2 protein,BMP4 protein and BMP7 protein are available from, for example, R&DSystems, and GDF7 protein is available from, for example, Wako PureChemical Industries, Ltd.

The concentration of a substance acting on the BMP signal transductionpathway only needs to be a concentration capable of inducingdifferentiation of cells forming pluripotent stem cell aggregates intoretinal cells. In the case of BMP4, for example, it is added to a mediumat a concentration of about 0.01 nM to about 1 pM, preferably about 0.1nM to about 100 nM, more preferably about 1.5 nM.

A substance acting on the BMP signal transduction pathway only needs tobe added after about 24 hours from the start of the floating culture instep (1), and may be added to the medium within several days from thestart of the floating culture (e.g., within 15 days). Preferably, asubstance acting on the BMP signal transduction pathway is added to themedium between day 1 and day 15, more preferably between day 1 and day9, most preferably at day 3, from the start of the floating culture.

After a substance acting on the BMP signal transduction pathway is addedto the medium and differentiation induction of cells forming pluripotentstem cell aggregates into retinal cells is started, the substance actingon the BMP signal transduction pathway does not need to be added to themedium, and the medium can be exchanged with a serum-free medium orserum-containing medium each being free of a substance acting on the BMPsignal transduction pathway, whereby the cost of the medium can besuppressed. A cell in which differentiation induction into a retinalcell has been started can be confirmed, for example, by detecting theexpression of Rax gene in the cell. It is also possible to confirm thetime when the differentiation induction into retinal cell was started bysubjecting the aggregates formed in step (1) by using a pluripotent stemcell, in which a fluorescence reporter protein gene such as GFP has beenknocked-in in the Rax gene locus, to floating culture in the presence ofa substance acting on the BMP signal transduction pathway at aconcentration necessary for differentiation induction into retinalcells, and detecting the fluorescence emitted from the expressedfluorescence reporter protein. One of the embodiments of step (2) is astep including subjecting the aggregates formed in step (1) to floatingculture in a serum-free medium or serum-containing medium eachcontaining a substance acting on the BMP signal transduction pathway ata concentration necessary for differentiation induction into retinalcells and being free of a substance acting on the Sonic hedgehog signaltransduction pathway until a cell expressing the Rax gene appears toobtain an aggregate containing a retinal progenitor cell.

The culture conditions such as culture temperature, CO₂ concentration instep (2) can be appropriately determined. The culture temperature is,for example, about 30 to about 40° C., preferably about 37° C. The CO₂concentration is, for example, about 1 to about 10%, preferably about5%.

That an aggregate containing a retinal progenitor cell has been obtainedcan be confirmed by, for example, detecting that the aggregate containsa cell expressing Rax or PAX6, which is a retinal progenitor cellmarker.

The obtained aggregate containing a retinal progenitor cell may be usedas it is as a reagent for evaluating toxicity or drug efficacy. It isalso possible to obtain a highly pure retinal progenitor cell bysubjecting the aggregate containing a retinal progenitor cell to adispersion treatment (e.g., trypsin/EDTA treatment), and selecting theobtained cells by using FACS.

The production method 2 of the present invention is a method forproducing a retinal tissue, comprising the following steps (1), (2) and(3):

(1) a first step of subjecting pluripotent stem cells to floatingculture in a serum-free medium to form an aggregate of pluripotent stemcells,(2) a second step of subjecting the aggregate formed in step (1) tofloating culture in a serum-free medium or serum-containing medium eachbeing free of a substance acting on the Sonic hedgehog signaltransduction pathway and containing a substance acting on the BMP signaltransduction pathway, thereby obtaining an aggregate containing retinalprogenitor cells, and(3) a third step of subjecting the aggregate formed in step (2) tofloating culture in a serum-free medium or serum-containing medium eachbeing free of any of a substance acting on the Sonic hedgehog signaltransduction pathway, a substance acting on the BMP signal transductionpathway and a substance acting on the Wnt signal pathway, therebyobtaining an aggregate containing retinal tissues and beingsubstantially free of non-neural head ectoderm.

The step (1) and step (2) of the production method 2 of the presentinvention can be performed similarly to step (1) and step (2) of theproduction method 1 of the present invention.

The step (3) including subjecting the aggregate formed in step (2) tofloating culture in a serum-free medium or serum-containing medium eachbeing free of any of a substance acting on the Sonic hedgehog signaltransduction pathway, a substance acting on the BMP signal transductionpathway and a substance acting on the Wnt signal pathway, therebyobtaining an aggregate containing retinal tissues and beingsubstantially free of non-neural head ectoderm is explained.

The medium used in step (3) is, for example, a serum-free medium orserum-containing medium not supplemented with any of a substance actingon the Sonic hedgehog signal transduction pathway, a substance acting onthe BMP signal transduction pathway and a substance acting on the Wntsignal pathway.

The medium “free of any of a substance acting on the Sonic hedgehogsignal transduction pathway, a substance acting on the BMP signaltransduction pathway and a substance acting on the Wnt signal pathway”also includes a medium substantially free of any of a substance actingon the Sonic hedgehog signal transduction pathway, a substance acting onthe BMP signal transduction pathway and a substance acting on the Wntsignal pathway, for example, a medium not containing any of a substanceacting on the Sonic hedgehog signal transduction pathway, a substanceacting on the BMP signal transduction pathway and a substance acting onthe Wnt signal pathway, at a concentration exerting an adverse influenceon the selective differentiation into retinal tissues.

The medium “not supplemented with any of a substance acting on the Sonichedgehog signal transduction pathway, a substance acting on the BMPsignal transduction pathway and a substance acting on the Wnt signalpathway” also includes a medium substantially not supplemented with anyof a substance acting on the Sonic hedgehog signal transduction pathway,a substance acting on the BMP signal transduction pathway and asubstance acting on the Wnt signal pathway, for example, a medium notsupplemented with any of a substance acting on the Sonic hedgehog signaltransduction pathway, a substance acting on the BMP signal transductionpathway and a substance acting on the Wnt signal pathway, at aconcentration exerting an adverse influence on the selectivedifferentiation into retinal tissues.

The serum-free medium or serum-containing medium to be used as suchmedium is not particularly limited as long as it is as mentioned above.To avoid complicated formulation process, for example, a serum-freemedium supplemented with an appropriate amount of a serum replacementsuch as commercially available KSR (e.g., a medium obtained by adding10% KSR, 450 μM 1-monothioglycerol and 1×Chemically Defined LipidConcentrate to a 1:1 mixture of IMDM and F-12) is preferably used. Theamount of KSR to be added to a serum-free medium is generally about 1%to about 20%, preferably about 2% to about 20%, in the case of, forexample, human ES cells.

The substance acting on the Wnt signal pathway is a substance that canenhance signal transduction mediated by Wnt. Examples of the substanceacting on the Wnt signal pathway include protein belonging to Wnt family(e.g., Wnt1, Wnt3a, Wnt7a), Wnt receptor, Wnt receptor agonist, GSK3βinhibitor (e.g., 6-Bromoindirubin-3′-oxime (BIO), CHIR99021,Kenpaullone) and so on.

The culture conditions such as culture temperature, CO₂ concentration,O₂ concentration in step (3) can be appropriately determined. Theculture temperature is, for example, about 30° C. to about 40° C.,preferably about 37° C. The CO₂ concentration is, for example, about 1to about 10%, preferably about 5%. The O₂ concentration is not less thanabout 18%, for example, about 20% to about 70%, preferably about 20% toabout 60%, more preferably about 20% to about 50%.

While the culture period in step (3) is not particularly limited, it isgenerally 48 hr or longer, preferably 7 days or longer.

In the aggregates subjected to floating culture in such manner, theretinal tissue is present to cover the surface of the aggregate. Theretinal tissue can be confirmed by, after completion of the floatingculture, fixing the aggregates with a fixative such as para-formaldehydesolution, preparing a frozen section, and confirming formation of aretinal tissue having a layer structure by an immunostaining method andso on. Since respective layers of a retinal tissue are composed ofdifferent retinal progenitor cells (photoreceptor, horizontal cell,bipolar cell, amacrine cell, retinal ganglion cell), formation of alayer structure can be confirmed by an immunostaining method usingantibodies against the markers expressed in these cells.

It is also possible to physically cut out the retinal tissue present onthe surface of aggregates with tweezers and so on. In this case, since aneural tissue other than a retinal tissue may be formed on the surfaceof each aggregate, a part of the neural tissue cut out from theaggregate is severed and confirmed by the immunostaining method asdescribed below and so on, whereby the tissue is confirmed to be aretinal tissue.

In an aggregate containing the retinal tissue and substantially free ofnon-neural head ectoderm, for example, RAX positive tissues are observedin the immunostained images of the aggregate cryosection, and RAXnegative tissues are not observed in the outside thereof.

Retinal tissues can be obtained highly efficiently from humanpluripotent stem cells by the production method 2 of the presentinvention. Since the retinal tissues obtained by the production method 2of the present invention contain a neuron specific to each of the retinalayers, it is also possible to obtain a cell constituting retinaltissues such as photoreceptor, horizontal cell, bipolar cell, amacrinecell, ganglion cell, or progenitor cells of these and the like. Whatcell was available from the obtained retinal tissue can be confirmed bya method known per se, for example, expression of a cell marker.

An aggregate containing the obtained retinal tissue and substantiallyfree of non-neural head ectoderm may be used as it is as a reagent forevaluating toxicity or drug efficacy. It is also possible to obtain ahighly pure retinal tissue constituting cell by subjecting the aggregatecontaining a retinal tissue and substantially free of non-neural headectoderm to a dispersion treatment (e.g., trypsin/EDTA treatment), andselecting the obtained cells by using FACS.

The production method 3 of the present invention is a method forproducing a retinal layer-specific neural cell, comprising the followingsteps (1), (2) and (3):

(1) a first step of subjecting pluripotent stem cells to floatingculture in a serum-free medium to form an aggregate of pluripotent stemcells,(2) a second step of subjecting the aggregate formed in step (1) tofloating culture in a serum-free medium or serum-containing medium eachbeing free of a substance acting on the Sonic hedgehog signaltransduction pathway and containing a substance acting on the BMP signaltransduction pathway, thereby obtaining an aggregate containing retinalprogenitor cells, and(3) a third step of subjecting the aggregate formed in step (2) tofloating culture in a serum-free medium or serum-containing medium eachbeing free of any of a substance acting on the Sonic hedgehog signaltransduction pathway, a substance acting on the BMP signal transductionpathway and a substance acting on the Wnt signal pathway until theintended retinal layer-specific neural cells appear, thereby obtainingan aggregate containing retinal tissues containing the intended retinallayer-specific neural cells and being substantially free of non-neuralhead ectoderm.

The step (1) and step (2) of the production method 3 of the presentinvention can be performed similarly to step (1) and step (2) of theproduction method 1 of the present invention.

The step (3) including subjecting the aggregate formed in step (2) tofloating culture in a serum-free medium or serum-containing medium eachbeing free of any of a substance acting on the Sonic hedgehog signaltransduction pathway, a substance acting on the BMP signal transductionpathway and a substance acting on the Wnt signal pathway until theintended retinal layer-specific neural cells appear, thereby obtainingan aggregate containing retinal tissues containing the intended retinallayer-specific neural cells and being substantially free of non-neuralhead ectoderm is explained.

The medium used in step (3) is, for example, a serum-free medium orserum-containing medium not supplemented with any of a substance actingon the Sonic hedgehog signal transduction pathway, a substance acting onthe BMP signal transduction pathway and a substance acting on the Wntsignal pathway.

The medium “free of any of a substance acting on the Sonic hedgehogsignal transduction pathway, a substance acting on the BMP signaltransduction pathway and a substance acting on the Wnt signal pathway”also includes a medium substantially free of any of a substance actingon the Sonic hedgehog signal transduction pathway, a substance acting onthe BMP signal transduction pathway and a substance acting on the Wntsignal pathway, for example, a medium not containing any of a substanceacting on the Sonic hedgehog signal transduction pathway, a substanceacting on the BMP signal transduction pathway and a substance acting onthe Wnt signal pathway, at a concentration exerting an adverse influenceon the selective differentiation into retinal tissues and retinallayer-specific neural cells.

The medium “not supplemented with any of a substance acting on the Sonichedgehog signal transduction pathway, a substance acting on the BMPsignal transduction pathway and a substance acting on the Wnt signalpathway” also includes a medium substantially not supplemented with anyof a substance acting on the Sonic hedgehog signal transduction pathway,a substance acting on the BMP signal transduction pathway and asubstance acting on the Wnt signal pathway, for example, a medium notsupplemented with any of a substance acting on the Sonic hedgehog signaltransduction pathway, a substance acting on the BMP signal transductionpathway and a substance acting on the Wnt signal pathway, at aconcentration exerting an adverse influence on the selectivedifferentiation into retinal tissues and retinal layer-specific neuralcells.

The serum-free medium or serum-containing medium to be used as suchmedium is not particularly limited as long as it is as mentioned above.For example, a serum-containing medium obtained by adding 10% fetal calfserum, N2 supplement, 100 μM taurine, and 500 nM retinoic acid toDMEM-F12 medium, a serum-free medium supplemented with an appropriateamount of a serum replacement such as commercially available KSR (e.g.,a medium obtained by adding 10% KSR, 450 μM 1-monothioglycerol and 1 xChemically Defined Lipid Concentrate to a 1:1 mixture of IMDM and F-12)and the like can be mentioned.

The culture conditions such as culture temperature, CO₂ concentration,O₂ concentration in step (3) can be appropriately determined. Theculture temperature is, for example, about 30° C. to about 40° C.,preferably about 37° C. The CO₂ concentration is, for example, about 1%to about 10%, preferably about 5%. The O₂ concentration is not less thanabout 18%, for example, about 20% to about 70%, preferably about 20% toabout 60%, more preferably about 20% to about 50%.

The culture period in step (3) varies depending on the intended retinallayer-specific neural cell and is, for example, about 7 days to about200 days.

In the aggregate subjected to floating culture in such manner, theretinal tissue containing the retinal layer-specific neural cell ispresent to cover the surface of the aggregate. The retinal tissue can beconfirmed by, after completion of the floating culture, fixing theaggregates with a fixative such as para-formaldehyde solution, preparinga frozen section, and confirming formation of a retinal tissue having alayer structure by an immunostaining method and so on. Since respectivelayers of a retinal tissue are composed of different retinal progenitorcells (photoreceptor, horizontal cell, bipolar cell, amacrine cell,retinal ganglion cell), formation of a layer structure can be confirmedby an immunostaining method using antibodies against the markersexpressed in these cells.

In an aggregate containing the retinal tissue containing a retinallayer-specific neural cell and substantially free of non-neural headectoderm, for example, RAX positive tissues are observed in theimmunostained images of the aggregate cryosection, and RAX negativetissues are not observed in the outside thereof.

Since the retinal tissues obtained by the production method 3 of thepresent invention contain a neuron specific to each of the retinalayers, it is also possible to obtain a retinal layer-specific neuralcell such as photoreceptor, horizontal cell, bipolar cell, amacrine celland ganglion cell. What cell was available from the obtained retinaltissue can be confirmed by a method known per se, for example,expression of a cell marker.

An aggregate containing the obtained retinal tissue containing retinallayer-specific neural cell and substantially free of non-neural headectoderm may be used as it is as a reagent for evaluating toxicity ordrug efficacy. It is also possible to obtain a highly pure retinallayer-specific neural cell by subjecting the aggregate containing aretinal tissue containing retinal layer-specific neural cell andsubstantially free of non-neural head ectoderm to a dispersion treatment(e.g., trypsin/EDTA treatment), and selecting the obtained cells byusing FACS.

The obtained retinal layer-specific neural cells can be directlycultured furthermore, or subjected to a dispersion treatment (e.g.,trypsin/EDTA treatment) and further culture under adhesion conditions.In the case of adhesion culture, a cell adhesive culture vessel, forexample, a culture vessel after a coating treatment with anextracellular matrix etc. (e.g., poly-D-lysine, laminin, fibronectin),is preferably used. The culture conditions of the adhesion culture suchas culture temperature, CO₂ concentration, and O₂ concentration can bedetermined as appropriate. In this case, culture may be performed in thepresence of a known differentiation induction substance and aneurotrophic factor. Examples of the differentiation induction substanceand neurotrophic factor include NGF (Biochem. Biophys. Res. Commun.,199, 552 (1994)), retinoic acid (Dev. Biol., 168, 342 (1995); J.Neurosci., 16, 1056 (1996)), BMP inhibitory factor (Nature, 376, 333-336(1995)), IGF (Genes&Development, 15, 3023-8 (2003)), BDNF, NT3, NT4 andso on.

The produced retinal tissues and retinal layer-specific neural cells canbe confirmed using the presence or absence of expression of a cellmarker and the like as indices, or by combining them as necessary. Theobtained retinal layer-specific neural cell can also be identified bythe observation of the cell morphology. A desired, particular cell canalso be isolated based on such marker expression pattern and cellmorphology.

The retinal progenitor cell, retinal tissue or retinal layer-specificneural cell produced by the production methods 1 to 3 of the presentinvention can also be used for screening for a therapeutic drug for adisease due to a disorder of retinal tissue or retina-related cell, or atransplantation material for cell treatment, a material for the study ofdiseases or a drug discovery material for a therapeutic drug for a celldamage due to other etiology. In addition, they can be utilized for thestudy, test and so on of such toxicity as phototoxicity in the toxicityand drug efficacy evaluation of chemical substances and so on.

Examples of the disease due to a disorder of retinal tissue orretina-related cell include organic mercury poisoning, chloroquineretinopathy, retinitis pigmentosa, age-related macular degeneration,glaucoma, diabetic retinopathy, neonatal retinopathy, and so on.

The retinal progenitor cell, retinal tissue or retinal layer-specificneural cell produced by the production methods 1 to 3 the presentinvention can be used as retina for transplantation, which is used forsupplementing a damaged cell or disordered tissue itself in a celldamage state (e.g., used for transplantation operation) and so on.

EXAMPLES

The present invention is explained in more detail in the following byreferring to Examples, which are not to be s construed as limitative.

Example 1: Production Example of Aggregate Containing Retinal ProgenitorCells

RAX::GFP knock-in human ES cells (KhES-1-derived; Cell Stem Cell, 2012,10(6) 771-785) were cultured according to the methods described in“Ueno, M. et al. PNAS 2006, 103(25), 9554-9559” and “Watanabe, K. et al.Nat Biotech 2007, 25, 681-686”. As the medium, a medium obtained byadding 20% KSR (KNOCKOUT™ Serum Replacement; Invitrogen), 0.1 mM2-mercaptoethanol, 2 mM L-glutamine, 1× non-essential amino acid, 5ng/ml bFGF to DMEM/F12 medium (Sigma) was used. The cultured ES cellswere dispersed into single cells by using TrypLE Express (Invitrogen),and the singly dispersed ES cells were suspended in 100 μl of aserum-free medium at 1.2×10⁴ cells per one well of a non-cell adhesive96 well culture plate (sumilon spheroid plates, SUMITOMO BAKELITE Co.,Ltd.) and subjected to floating culture at 37° C., 5% CO₂. As theserum-free medium therefor, a serum-free medium obtained by adding 10%KSR, 450 μM 1-monothioglycerol, 1x Chemically defined lipid concentrate,20 μM Y27632 to a 1:1 mixture of F-12 medium and IMDM medium was used.At day 3 from the start of the floating culture, any of humanrecombinant BMP4 (R&D) (final concentration 1.5 nM), BMP2 (R&D) (finalconcentration 100 ng/ml), BMP7 (R&D) (final concentration 100 ng/ml) andGDF7 (R&D) (final concentration 100 ng/ml) was added and floatingculture was performed. Similar culture was also performed underconditions free of the addition of any of the substances acting on theBMP signal transduction pathway. A half amount of the culture medium inthe well was exchanged every 3 days with the above-mentioned medium notsupplemented with any of substance acting on the BMP signal transductionpathway. At day 18 from the start of the floating culture, fluorescencemicroscopic observation was performed. When cultured under conditionswithout addition of a substance acting on the BMP signal transductionpathway, GFP expressing cells indicating induction of retinal progenitorcells were scarcely observed (FIG. 1A, B). In contrast, GFP expressingcells clearly increased under culture conditions including addition ofany of BMP2 (FIG. 1C, D), BMP4 (FIG. 1E, F), BMP7 (FIG. 1G, H), and GDF7(FIG. 1I, J).

Example 2: Production Example of retinal tissue—1

RAX::GFP knock-in human ES cells (KhES-1-derived; Cell Stem Cell, 2012,10(6) 771-785) were cultured according to the methods described in“Ueno, M. et al. PNAS 2006, 103(25), 9554-9559” and “Watanabe, K. et al.Nat Biotech 2007, 25, 681-686”. As the medium, a medium obtained byadding 20% KSR (KNOCKOUT· Serum Replacement; Invitrogen), 0.1 mM2-mercaptoethanol, 2 mM L-glutamine, lx non-essential amino acid, 5ng/ml bFGF to DMEM/F12 medium (Sigma) was used. The cultured ES cellswere dispersed into single cells by using TrypLE Express (Invitrogen),and the singly dispersed ES cells were suspended in 100 μl of aserum-free medium at 1.2×10⁴ cells per one well of a non-cell adhesive96 well culture plate (sumilon spheroid plates, SUMITOMO BAKELITE Co.,Ltd.) and subjected to floating culture at 37° C., 5% CO₂. As theserum-free medium therefor, a serum-free medium obtained by adding 10%KSR, 450 μM 1-monothioglycerol, 1× Chemically defined lipid concentrate,20 μM Y27632 to a 1:1 mixture of F-12 medium and IMDM medium was used.Human recombinant BMP4 (R&D) (final concentration 1.5 nM) was added atany time point from simultaneously with the start of the floatingculture, day 1 from the start of the floating culture, day 2 from thestart of the floating culture, and day 3 from the start of the floatingculture, and floating culture was performed. Similar culture was alsoperformed under conditions free of the addition of a substance acting onthe BMP signal transduction pathway. A half amount of the culture mediumin the well was exchanged every 3 days with the above-mentioned mediumnot supplemented with a substance acting on the BMP signal transductionpathway. At day 18 from the start of the floating culture, aggregateswere transferred from the 96 well plate to a floating culture dish, andfloating culture was continuously performed in a serum-free mediumobtained by adding 10% KSR, 450 μM 1-monothioglycerol, lx Chemicallydefined lipid concentrate to a 1:1 mixture of F-12 medium and IMDMmedium. At day 26 from the start of the floating culture, fluorescencemicroscopic observation and measurement of the GFP positive cells byFACS were conducted.

GFP expressing cell indicating induction of retinal progenitor cells wasnot found under conditions without addition of a substance acting on theBMP signal transduction pathway (FIG. 2A, B, C) and under conditionswith the addition of BMP4 simultaneously with the start of the floatingculture (FIG. 2D, E, F). In contrast, GFP expressing cell clearlyincreased under conditions with the addition of BMP4 at any time pointfrom day 1 from the start of the floating culture (FIG. 2G, H, I), day 2from the start of the floating culture (FIG. 2J, K, L), and day 3 fromthe start of the floating culture (FIG. 2M, N, O). In all conditions,RAX::GFP negative tissue was not observed on the outside of the RAX::GFPpositive tissue formed. According to the measurement results by FACS,not less than 85% of the cells were GFP positive under conditions withthe addition of BMP4 at day 3 from the start of the floating culture(FIG. 20). The above results reveal that addition of a substance actingon the BMP signal transduction pathway at day 1 or later, preferably atday 3, from the start of the floating culture is effective for theproduction of an aggregate containing retinal tissue and substantiallyfree of non-neural head ectoderm.

Example 3: Production Example of retinal tissue—2

RAX::GFP knock-in human ES cells (KhES-1-derived; Cell Stem Cell, 2012,10(6) 771-785) were cultured according to the methods described in“Ueno, M. et al. PNAS 2006, 103(25), 9554-9559” and “Watanabe, K. et al.Nat Biotech 2007, 25, 681-686”. As the medium, a medium obtained byadding 20% KSR (KNOCKOUT™ Serum Replacement; Invitrogen), 0.1 mM2-mercaptoethanol, 2 mM L-glutamine, 1× non-essential amino acid, 5ng/ml bFGF to DMEM/F12 medium (Sigma) was used. The cultured ES cellswere dispersed into single cells by using TrypLE Express (Invitrogen),and the singly dispersed ES cells were suspended in 100 μl of aserum-free medium at 1.2×10⁴ cells per one well of a non-cell adhesive96 well culture plate (sumilon spheroid plates, SUMITOMO BAKELITE Co.,Ltd.) and subjected to floating culture at 37° C., 5% CO₂. As theserum-free medium therefor, a serum-free medium obtained by adding 10%KSR, 450 μM 1-monothioglycerol, 1× Chemically defined lipid concentrate,20 μM Y27632 to a 1:1 mixture of F-12 medium and IMDM medium was used.Human recombinant BMP4 (R&D) (final concentration 1.5 nM) was added atany time point from day 6 from the start of the floating culture, day 9from the start of the floating culture, day 12 from the start of thefloating culture and day 15 from the start of the floating culture, andfloating culture was performed. Similar culture was also performed underconditions free of the addition of a substance acting on the BMP signaltransduction pathway. A half amount of the culture medium in the wellwas exchanged every 3 days with the above-mentioned medium notsupplemented with a substance acting on the BMP signal transductionpathway. At day 18 from the start of the floating culture, aggregateswere transferred from the 96 well plate to a floating culture dish, andfloating culture was continuously performed in a serum-free mediumobtained by adding 10% KSR, 450 μM 1-monothioglycerol, 1× Chemicallydefined lipid concentrate to a 1:1 mixture of F-12 medium and IMDMmedium. At day 24 from the start of the floating culture, fluorescencemicroscopic observation was conducted.

As a result, GFP expressing cell indicating induction of retinalprogenitor cells was not found under conditions without addition of asubstance acting on the BMP signal transduction pathway (FIG. 3A, B). Incontrast, GFP expressing cell clearly increased under conditions withthe addition of BMP4 at any time point from day 6 from the start of thefloating culture (FIG. 3C, D), day 9 from the start of the floatingculture (FIG. 3E, F), and day 12 from the start of the floating culture(FIG. 3G, H). Under conditions with the addition of BMP4 at day 15 fromthe start of the floating culture, GFP expressing cells were induced butthe efficiency was low (FIG. 31, J). In all conditions, RAX::GFPnegative tissue was not observed on the outside of the RAX::GFP positivetissue formed. The above results reveal that addition of a substanceacting on the BMP signal transduction pathway on or before day 15 fromthe start of the floating culture is effective for the production of anaggregate containing retinal tissue and substantially free of non-neuralhead ectoderm.

Example 4: Production Example of Retinal Tissue—3

At day 18 from the start of the floating culture, the aggregatescontaining GFP expressing cell, which were obtained in Example 1, weretransferred from the 96 well plate to a floating culture dish, andfloating culture was continuously performed in a serum-free mediumobtained by adding 10% KSR, 450 μM 1-monothioglycerol, 1× Chemicallydefined lipid concentrate to a 1:1 mixture of F-12 medium and IMDMmedium. At day 26 from the start of the floating culture, the aggregateswere fixed with 4% para-formaldehyde solution, cryosection was prepared,and the tissue structure was confirmed by the immunostaining method.

At day 26 from the start of the floating culture, all layers wereRAX::GFP positive, and the outer layer was positive to Chx10, which is aretina stem cell marker (FIG. 4A, B, C). No tissue such as nonneuralectoderm was present on the outside of the RAX::GFP positive epithelium.It was shown that the production method of the present invention canproduce retinal tissue highly efficiently from human ES cells.

Example 5: Production Example of Retinal Layer-Specific Neural Cell

RAX::GFP knock-in human ES cells (KhES-1-derived; Cell Stem Cell, 2012,10(6) 771-785) were cultured according to the methods described in“Ueno, M. et al. PNAS 2006, 103(25), 9554-9559” and “Watanabe, K. et al.Nat Biotech 2007, 25, 681- 686”. As the medium, a medium obtained byadding 20% KSR (KNOCKOUT™ Serum Replacement; Invitrogen), 0.1 mM2-mercaptoethanol, 2 mM L-glutamine, 1× non-essential amino acid, 5ng/ml bFGF to DMEM/F12 medium (Sigma) was used. The cultured ES cellswere dispersed into single cells by using TrypLE Express (Invitrogen),and the singly dispersed ES cells were suspended in 100 μl of aserum-free medium at 1.2×10⁴ cells per one well of a non-cell adhesive96 well culture plate (sumilon spheroid plates, SUMITOMO BAKELITE Co.,Ltd.) and subjected to floating culture at 37° C., 5% CO₂. As theserum-free medium therefor, a serum-free medium obtained by adding 10%KSR, 450 μM 1-monothioglycerol, 1× Chemically defined lipid concentrate,20 μM Y27632 to a 1:1 mixture of F-12 medium and IMDM medium was used.At day 3 from the start of the floating culture, human recombinant BMP4(R&D) (final concentration 1.5 nM) was added. A half amount of theculture medium in the well was exchanged every 3 days with theabove-mentioned medium not supplemented with a substance acting on theBMP signal transduction pathway. At day 18 from the start of thefloating culture, aggregates were transferred from the 96 well plate toa floating culture dish, and floating culture was continuously performedin a medium obtained by adding 10% fetal calf serum, N2 supplement, 100μM taurine, 500 nM retinoic acid to DMEM-F12 medium. From day 18 fromthe start of the floating culture, culture was performed under 40% O₂.At day 117 from the start of the floating culture, the aggregates werefixed with 4% para-formaldehyde solution, cryosection was prepared, andthe tissue structure was confirmed by the immunostaining method. At day117 from the start of the floating culture, all layers were RAX::GFPpositive, and a cell positive to Recoverin, which is a photoreceptormarker, was present (FIG. 5A). In addition, outer layer of the retinaltissue contained a cell positive to Nrl, which is a rod photoreceptormarker (FIG. 5B), and a cell positive to RXR-gamma, which is a conephotoreceptor marker (FIG. 5C), which suggests occurrence ofdifferentiation into rod, cone photoreceptors. Furthermore, a cellpositive to Chx10, which is a marker of retinal progenitor cell andbipolar cell (FIG. 5D), a cell positive to Calretinin, which is anamacrine cell marker (FIG. 5E), and a cell positive to Calbindin, whichis a horizontal cell marker (FIG. 5F) were present. The results revealthat the production method of the present invention can produce retinaltissue, which is constituted of various kinds of differentiated retinallayer-specific neural cells, highly efficiently from human ES cells.

Example 6: Production Example of Retinal Layer-Specific Neural Cell

RAX::GFP knock-in human ES cells (KhES-1-derived; Cell Stem Cell, 2012,10(6) 771-785) were cultured according to the methods described in“Ueno, M. et al. PNAS 2006, 103(25), 9554-9559” and “Watanabe, K. et al.Nat Biotech 2007, 25, 681-686”. As the medium, a medium obtained byadding 20% KSR (KNOCKOUT™ Serum Replacement; Invitrogen), 0.1 mM2-mercaptoethanol, 2 mM L-glutamine, 1× non-essential amino acid, 8ng/ml bFGF to DMEM/F12 medium (Sigma) was used. The cultured ES cellswere dispersed into single cells by using TrypLE Express (Invitrogen),and the singly dispersed ES cells were suspended in 100 μl of aserum-free medium at 1.2×10⁴ cells per one well of a non-cell adhesive96 well culture plate (sumilon spheroid plates, SUMITOMO BAKELITE Co.,Ltd.) and subjected to floating culture at 37° C., 5% CO₂. As theserum-free medium therefor, a serum-free medium obtained by adding 10%KSR, 450 μM 1-monothioglycerol, 1× Chemically defined lipid concentrate,20 μM Y27632 to a 1:1 mixture of F-12 medium and IMDM medium was used.At day 3 from the start of the floating culture, 50 μl of the serum-freemedium was added (total 150 μl). At day 6 from the start of the floatingculture, human recombinant BMP4 (R&D) (final concentration 1.5 nM) wasadded. A half amount of the culture medium in the well was exchangedevery 3 days with the above-mentioned medium not supplemented with asubstance acting on the BMP signal transduction pathway. At day 18 fromthe start of the floating culture, aggregates were transferred from the96 well plate to a floating culture dish, and floating culture wascontinuously performed in a medium obtained by adding 10% fetal calfserum, N2 supplement, 100 μM taurine, 500 nM retinoic acid to DMEM-F12medium. From day 18 from the start of the floating culture, culture wasperformed under 40% O₂. At day 50 from the start of the floatingculture, the aggregates were fixed with 4% para-formaldehyde solution,cryosection was prepared, and the tissue structure was confirmed by theimmunostaining method. At day 50 from the start of the floating culture,all layers were RAX::GFP positive, and a cell positive to Chx10, whichis a marker of retinal progenitor cell and bipolar cell (FIG. 6A) and acell positive to Pax6, which is a marker of ganglion cell and neuralcell (FIG. 6B) were present, which suggests formation of multi-layerretinal neural tissues. In addition, a cell positive to Crx, which is aphotoreceptor marker (FIG. 6C), and a cell positive to Brn3b, which is aganglion cell marker (FIG. 6D) were present. The results reveal that theproduction method of the present invention can produce retinal tissue,which is constituted of various kinds of differentiated retinallayer-specific neural cells, highly efficiently from human ES cells.

Example 7: Production Example of Aggregate Containing Retinal ProgenitorCell, Retinal Tissue and Retinal Layer-Specific Neural Cell, fromInduced Pluripotent Stem Cell (iPS Cell)

Human iPS cell line 201B7 (available from RIKEN BioResource center oriPS Academia Japan Inc.) is cultivated according to the methodsdescribed in “Deno, M. et al. PNAS 2006, 103(25), 9554-9559” and“Watanabe, K. et al. Nat Biotech 2007, 25, 681-686”. As the medium, amedium obtained by adding 20% KSR (KNOCKOUT™ Serum Replacement;Invitrogen), 0.1 mM 2-mercaptoethanol, 2 mM L-glutamine, 1×non-essential amino acid, 5 ng/ml bFGF to DMEM/F12 medium (Sigma) isused. The cultured iPS cells are dispersed into single cells by usingTrypLE Express (Invitrogen), suspended in 100 μl of a serum-free mediumat 1.2×10⁴ cells per one well of a non-cell adhesive 96 well cultureplate (sumilon spheroid plates, SUMITOMO BAKELITE Co., Ltd.) andsubjected to floating culture at 37° C., 5% CO₂. As the serum-freemedium therefor, a serum-free medium obtained by adding 10% KSR, 450 μM1-monothioglycerol, 1× Chemically defined lipid concentrate, 20 μMY27632 to a 1:1 mixture of F-12 medium and IMDM medium is used. At sometime point between day 1 and day 15 from the start of the floatingculture, any of human recombinant BMP4 (R&D) (final concentration 1.5nM), BMP2 (R&D) (final concentration 100 ng/ml), BMP7 (R&D) (finalconcentration 100 ng/ml), and GDF7 (R&D) (final concentration 100 ng/ml)is added and floating culture is performed. A half amount of the culturemedium in the well is exchanged every 3 days with the above-mentionedmedium not supplemented with any substance acting on the BMP signaltransduction pathway. At day 18 from the start of the floating culture,a part of the aggregates is recovered, fixed with 4% para-formaldehyde,cryosection is prepared, and expression of retinal progenitor cellmarkers (Rax, Chx10) is confirmed by the immunostaining method. The restof the aggregates is transferred from a 96 well plate to a floatingculture dish, and floating culture is continuously performed in aserum-free medium obtained by adding 10% fetal calf serum, N2supplement, 100 μM taurine, 500 nM retinoic acid to DMEM-F12 medium.From day 18 from the start of the floating culture, culture is performedunder 40% O₂. At day 117 from the start of the floating culture, theaggregates are fixed with 4% para-formaldehyde solution, cryosection isprepared, and the tissue structure and expression of a retinallayer-specific neural cell markers (Nrl, RXR-gamma, Recoverin, Chx10,Calretinin, Calbindin) are confirmed by the immunostaining method.

In this way, a retinal progenitor cell, and further, a retinal tissueconstituted of various kinds of differentiated retinal layer-specificneural cells can be produced from human iPS cells.

This application is based on a patent application No. 2013-173285 filedin Japan (filing date: Aug. 23, 2013), the contents of which areincorporated in full herein.

INDUSTRIAL APPLICABILITY

According to the production method of the present invention, a retinalprogenitor cell, a retinal tissue or a retinal layer-specific neuralcell can be produced with high efficiency. In the production method ofthe present invention, since a retinal progenitor cell, a retinal tissueor a retinal layer-specific neural cell can be obtained by floatingculture of an aggregate without adding a basement membrane preparationto a medium, the risk of contamination of the obtained cell or tissuewith a component derived from a heterologous species is reduced. Theproduction method of the present invention is highly useful since itefficiently produces a cell group (such as photoreceptor and opticnerve) constituting a retinal tissue, for the purpose of toxicity ordrug efficacy evaluation of a chemical substance, etc., a cell treatmentand so on, as well as efficiently produces a retinal tissue to be a“tissue material” to be used for tests and treatments for the purpose ofapplication to a toxicity or drug efficacy evaluation using a retinaltissue with a tissue structure, and to a transplantation material for aretinal tissue transplantation treatment.

1. A method for producing a retinal progenitor cell, comprising (1) afirst step of subjecting pluripotent stem cells to floating culture in aserum-free medium to form an aggregate of pluripotent stem cells, and(2) a second step of subjecting the aggregate formed in step (1) tofloating culture in a serum-free medium or serum-containing medium eachbeing free of a substance acting on the Sonic hedgehog (Shh) signaltransduction pathway that can enhance signal transduction mediated byShh at a concentration exerting an adverse influence on the selectivedifferentiation into retinal progenitor cell and retinal tissue andcontaining a substance acting on the bone morphogenic protein (BMP)signal transduction pathway that can enhance signal transduction pathwaymediated by BMP at a concentration necessary for differentiationinduction into retinal cells from day 1 or later from the start of thefloating culture in step (1) until a cell expressing retina and anteriorneural fold homeobox (Rax) gene appears, thereby obtaining an aggregatecontaining retinal progenitor cells.
 2. A method for producing a retinaltissue, comprising (1) a first step of subjecting pluripotent stem cellsto floating culture in a serum-free medium to form an aggregate ofpluripotent stem cells, (2) a second step of subjecting the aggregateformed in step (1) to floating culture in a serum-free medium orserum-containing medium each being free of a substance acting on theSonic hedgehog (Shh) signal transduction pathway that can enhance signaltransduction mediated by Shh at a concentration exerting an adverseinfluence on the selective differentiation into retinal progenitor celland retinal tissue and containing a substance acting on the bonemorphogenic protein (BMP) signal transduction pathway that can enhancesignal transduction pathway mediated by BMP at a concentration necessaryfor differentiation induction into retinal cells from day 1 or laterfrom the start of the floating culture in step (1) until a cellexpressing retina and anterior neural fold homeobox (Rax) gene appears,thereby obtaining an aggregate containing retinal progenitor cells, and(3) a third step of subjecting the aggregate formed in step (2) tofloating culture in a serum-free medium or serum-containing medium eachbeing free of any of a substance acting on the Shh signal transductionpathway that can enhance signal is transduction mediated by Shh, asubstance acting on the BMP signal transduction pathway that can enhancesignal transduction pathway mediated by BMP and a substance acting onthe Wingless-related integration site (Wnt) signal pathway that canenhance signal transduction mediated by Wnt, thereby obtaining anaggregate containing retinal tissues and being substantially free ofnon-neural head ectoderm.
 3. A method for producing a retinallayer-specific neural cell, comprising (1) a first step of subjectingpluripotent stem cells to floating culture in a serum-free medium toform an aggregate of pluripotent stem cells, (2) a second step ofsubjecting the aggregate formed in step (1) to floating culture in aserum-free medium or serum-containing medium each being free of asubstance acting on the Sonic hedgehog (Shh) signal transduction pathwaythat can enhance signal transduction mediated by Shh at a concentrationexerting an adverse influence on the selective differentiation intoretinal progenitor cell and retinal tissue and containing a substanceacting on the bone morphogenic protein (BMP) signal transduction pathwaythat can enhance signal transduction pathway mediated by BMP at aconcentration necessary for differentiation induction into retinal cellsfrom day 1 or later from the start of the floating culture in step (1)until a cell expressing retina and anterior neural fold homeobox (Rax)gene appears, thereby obtaining an aggregate containing retinalprogenitor cells, and (3) a third step of subjecting the aggregateformed in step (2) to floating culture in a serum-free medium orserum-containing medium each being free of any of a substance acting onthe Shh signal transduction pathway that can enhance signal transductionmediated by Shh, a substance acting on the BMP is signal transductionpathway that can enhance signal transduction pathway mediated by BMP anda substance acting on the Wingless-related integration site (Wnt) signalpathway that can enhance signal transduction mediated by Wnt until theintended retinal layer-specific neural cells appear, thereby obtainingan aggregate containing retinal tissues containing the intended retinallayer-specific neural cells and being substantially free of non-neuralhead ectoderm.
 4. The method according to claim 2, wherein thepluripotent stem cells are primate pluripotent stem cells.
 5. The methodaccording to claim 2, wherein the pluripotent stem cells are humanpluripotent stem cells.
 6. The method according to claim 2, wherein thefloating culture is performed in the absence of a basement membranepreparation.
 7. The method according to claim 2, wherein the substanceacting on the BMP signal transduction pathway that can enhance signaltransduction pathway mediated by BMP is one or more proteins selectedfrom the group consisting of BMP2, BMP4, BMP7 and GDF7.
 8. The methodaccording to claim 2, wherein the s substance acting on the BMP signaltransduction pathway that can enhance signal transduction pathwaymediated by BMP is BMP4.
 9. The method according to claim 8, whereinBMP4 is at a concentration of about 1.5 nM.
 10. The method according toclaim 2, wherein the substance acting on the BMP signal transductionpathway that can enhance signal transduction pathway mediated by BMP isadded to the medium between day 1 and day 15 from the start of thefloating culture in step (1).
 11. A method for producing a retinaltissue, comprising producing an aggregate containing retinal tissues andbeing substantially free of non-neural head ectoderm by the methodaccording to claim 2, and physically cutting out a retinal tissue fromthe thus-produced aggregate, thereby obtaining a purified retinaltissue.
 12. A method for producing a transplantation material for celltreatment, comprising producing an aggregate containing retinal tissuesand being substantially free of non-neural head ectoderm by the methodaccording to claim 2, and physically cutting out a retinal tissue fromthe aggregate to provide a transplantation material for cell treatment.13. A method for producing a transplantation material for celltreatment, comprising (1) producing an aggregate containing retinaltissues and being substantially free of non-neural head ectoderm by themethod according to claims 2, and (2) dispersing the aggregate producedin (1) and s selecting from the dispersed cells a retinal tissueconstituting cell to provide a transplantation material for celltreatment.
 14. A method of evaluating toxicity or drug efficacy of atest substance, comprising producing an aggregate containing retinaltissues and being substantially free of non-neural head ectoderm by themethod according to claim 2, and bringing thus-produced aggregate intocontact with the test substance, and examining the influence of thesubstance on the tissue in the aggregate.
 15. An in vitro cultured cellaggregate containing retinal tissues and being substantially free ofnon-neural head ectoderm, wherein not less than 85% of the cells are RAXpositive in the aggregate.
 16. A method for treating a disease due to adisorder of a retinal tissue, comprising transplanting an effectiveamount of a retinal tissue to a subject in need of the transplantation,wherein the retinal tissue is obtained by: (1) producing an aggregatecontaining retinal tissues and being substantially free of non-neuralhead ectoderm by the 25 method according to claims 2, and (2) physicallycutting out a retinal tissue from the aggregate produced in (1), therebyobtaining a purified retinal tissue.
 17. A method for treating a diseasedue to a disorder of a retinal tissue, comprising transplanting aneffective amount of a retinal tissue constituting cell to a subject inneed of the transplantation, wherein the retinal tissue constitutingcell is obtained by: (1) producing an aggregate containing retinaltissues and being substantially free of non-neural head ectoderm by themethod according to claims 2, and (2) dispersing the aggregate producedin (1) and selecting from the dispersed cells a retinal tissueconstituting cell, thereby obtaining a purified retinal tissueconstituting cell.